Stable avidin composition

ABSTRACT

Compositions and methods for avidin immobilized on an inert support material, e.g., agarose, are disclosed. The compositions have high activity levels of avidin and may further include a bulking agent, e.g., maltose, and a protectant to maintain the stability and integrity of the avidin agarose during lyophilization and terminal sterilization processes. These compositions have applicability in any instance where avidin agarose and/or the avidin/biotin technology are useful. In particular, the present compositions are useful in an enzyme capture system to prepare fibrin monomer useful for fibrin sealants.

BACKGROUND

The avidin-biotin affinity-based technology has found wide applicabilityin numerous fields of biology and biotechnology since the pioneeringwork by Dr. Edward Bayer and Dr. Meier Wilchek in the 1970's. Theaffinity constant between avidin and biotin is remarkably high and isnot significantly lessened when biotin is coupled to a wide variety ofbiomolecules. This affinity is substantially maintained even whenderivatized forms of the biotin are employed and numerous chemistrieshave been identified for coupling biomolecules to biotin with minimal ornegligible loss in the activity or other desired characteristics of thebiomolecule. Originally applied to purification and localizationprocedures for biologically active macromolecules, avidin-biotintechnology today has widespread use in medical diagnostics. Newerapplications which continue to be developed include affinity targeting,cell cytometry, blotting technology, drug delivery, hybridomatechnology, human stem cell selection and reinfusion as well as severalapproaches to enzyme capture. In some applications, avidin isimmobilized onto an inert material over which a solution containingbiotinylated biomolecules is passed. The affinity of the biotin for theavidin provides for the separation of the biomolecule from the solution.A review of the biotin-avidin technology can be found in Applications ofAvidin-Biotin Technology to Affinity-Based Separation, Bayer, et al., J.of Chromatography, 1990, pgs. 3-11.

EP 592242 describes a novel fibrin sealant based on fibrin monomer asopposed to the traditional fibrinogen-based sealants and involvessubjecting fibrinogen to a thrombin-like enzyme which is preferablyremoved after such treatment. EP 592242 describes that the enzymecapture and removal can be accomplished by using biotinylated batroxobinwhich can be recaptured with an avidin material. The fibrin monomersealant described in EP 592242 is advantageously completely autologous.Since autologous fibrin sealants can not always be prepared in advance,autologous processes which provide such sealants in short periods oftime (i.e., less than one hour or preferably less than 30 minutes) fromthe patients' own blood provide a great advantage over currenttechniques and products. The speed with which such autologous processescan be carried out is dependent to a large degree on the activity of thebiotin-and avidin-based reagents. Commercially available immobilizedavidin typically contains about 200 to 400 biotin binding units (BBU) ofactivity (where 1 BBU will bind 1 μg of α-biotin) per gram oflyophilized powder (e.g., avidin on acrylic beads from Sigma) or about20 to 50 BBU per milliliter of slurry or gel (e.g., avidin on agaroseavailable from Sigma and Pierce).

Also, the above fibrin monomer technology and other biologicalapplications would benefit from more convenient forms of avidin-andbiotin-based reagents. For example, the processing necessary to preparesuch compositions can have an adverse effect on the activity levelssince many of the coupling/immobilization techniques involve materialswhich can significantly reduce these activities. Additionally, systemswhich reduce or eliminate leaching of avidin or of the avidin-biotincomplexes would be advantageous in many applications. Further, manybiological applications would be greatly enhanced by the availability ofhigh activity avidin compositions which could be lyophilized andfurther, terminally sterilized while maintaining stability. Clearly,avidin compositions having higher avidin activity levels with greaterstability, especially in freeze dried powder forms capable ofwithstanding terminal sterilization, e.g., gamma irradiation, would bean advance in the art.

SUMMARY OF THE INVENTION

In accordance with the present invention, stable, highly activecompositions of avidin and an inert, easily separable support materialsuch as a water soluble polymer, e.g., polysaccharides selected fromagars and alginates, and having an activity level of 1000 BBU or moreper gram of lyophilized form and 50 BBU or more per milliliter of slurryor hydrated gel, are disclosed. Preferred compositions include a bulkingagent selected from nonionic water soluble polymers, a protectant, andthe avidin/inert support material. These compositions may also includeone or more materials selected to adjust and/or maintain the pH of thecomposition and are useful in an aqueous suspension or preferably inlyophilized form.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Although avidin/inert support compositions are known, such compositionshaving the high levels of activity as described in this invention havenot been heretofore disclosed. It has surprisingly been found thatavidin compositions can now be prepared having activity levels multiplesbeyond those presently available without damaging the integrity andstability of the avidin or its inert support. Indeed, these compositionsin any convenient form, i.e., slurry, suspension, hydrated gel,dehydrated gel, dried powders, etc., can be the basis of sterile, stableaqueous suspensions and remarkably stable lyophilized compositionscapable of withstanding terminal sterilization, e.g., gamma irradiation.The novel compositions herein have activity levels for lyophilized formsin excess of 1000 BBU (which is a measure of the biotin bindingcapability of the avidin composition as discussed above) and preferablybetween 1500 and 3000 BBU of activity and most preferably between 1800and 2400 BBU of activity per gram of powder. In slurry, suspension orgel forms the novel avidin compositions of the present invention haveactivity levels in excess of 50 BBU and preferably between 75 and 150BBU and more preferably between 90 and 110 BBU of activity permilliliter.

The preferred lyophilized avidin/inert support compositions of thepresent invention are stable, can be terminally sterilized, have lowmoisture uptake/moisture content, reswell completely and rapidly uponreconstitution, are non-leachable and pharmaceutically acceptable. Thebulking agents used in conjunction with this invention protect the gelbead from damage during the freeze drying process and provide for rapidand complete reswelling of the gel beads upon reconstitution of thefreeze dried material. The unique combination of components herein alsoprotects the avidin/inert support component from any deleterious effectsupon terminal sterilization of the composition. Thus, the affinity ofthe avidin for biotinylated biomolecules is maintained even after theserigorous processing steps, i.e., lyophilization and/or terminalsterilization. Accordingly, in the various applications where theaffinity characteristics of the avidin and the integrity of the inertsupport are both critical to the effectiveness of the avidin/biotintechnology, a superior composition is provided by the invention.

The avidin component of the present invention refers to avidin,monomeric avidin, Streptavidin, other proteins having an affinity forbiotin including derivatized forms of avidin and recombinant forms ofany of the above. The avidin should be insoluble, which is preferred, orotherwise easily separable from the inert support using techniques suchas emulsion/phase separation and the like. The inert support material isany material that has low reactivity, is hydrophilic, forms a porous ornonporous matrix which can be readily separated from a liquid phase ofthe reaction mixture when necessary. Such materials include, but are notlimited to, dextran, cellulose, starch, carageenan, chitin,polyacrylamide, hydroxyethylmethacrylate, stryrenedivinylbenzene,oxiraneacrylic, silica, alumina, zirconia, glass, perfluorocarbons andpolysaccharides from the agar family or the alginate family which formgel beads. These and other such materials are useful and well known asinert materials in separation columns. Most are commercially available,e.g., agarose which is preferred and which is available as Sepharose™from Pharmacia.

The invention will be further described referring to avidin and agarose.It should be understood that avidin is meant to include any of the formsof avidin described hereinabove and agarose represents any of the inertsupport materials described hereinabove.

The bulking agent is a nonionic water soluble polymer. Examples include,but are not limited to, simple sugars (e.g., mono- and di-saccharides),oligosaccharides, polysaccharides, polyvinylpyrrolidone,polyvinylalcohol or polyethyleneglycol. Preferably, the bulking agent isa sugar ranging in molecular weight from that of glucose up to andincluding that of high molecular weight dextran. More preferably, thebulking agent is an oligosaccharide based on glucose such as dimericglucose (i.e., maltose), trimeric glucose (maltotriose), maltotetraose,maltopentaose, maltohexaose, maltoheptaose, low molecular weightdextran, high molecular weight dextran including combinations of any ofthe above, with maltose being preferred.

The protectant of the present compositions is selected fromantioxidants, free radical scavengers and reducing agents. Preferred areantioxidants such as α-tocopherol, reduced glutathione, quinones, N,N-dimethyl-p-phenylenediamine, ascorbylpalmitate, amino acids, tartaricacid, phosphoric acid and ascorbic acid/sodium ascorbate with ascorbicacid/sodium, ascorbate being most preferred.

The present compositions may also include an agent to adjust the pH to adesired level. For example, alkaline materials, e.g., sodium hydroxidecan be added to adjust the pH which is preferably at about 4 for usewith biotinylated batroxobin. Further, buffers may be incorporated tomaintain the pH level. Buffers and agents to adjust the pH are wellknown in the art and any such materials are suitable depending upon theapplication. In a preferred embodiment, the ascorbic acid protectantalso serves as a buffer. However, it should be understood that anyconvenient buffer and pH can be utilized as required for the particularapplication.

The compositions of the present invention are conveniently in an aqueousslurry or suspension. Since these slurries or suspensions can either beprepared aseptically or can be terminally sterilized, they are also anintegral part of this invention. Preferably, the aqueous slurries orsuspensions of this invention are freeze dried since the lyophilizedpowders resulting therefrom are highly stable, terminally sterilizable(e.g., by gamma irradiation), non-hygroscopic and extremely easy tohandle.

As the compositions of this invention deal, inter alia, with beaded gelsin slurries or suspensions, it is important to clarify what some ofthese terms are understood to mean within this art. By way of example,agarose is commercially available in 4% and 6% gels. This refers to thefact that the hydrated gel bead material is, for example, 4% by weightof cross-linked agarose beads containing 96% by weight of water (i.e.,within the bead) for the 4% gel and 6% by weight of cross-linked agarosebeads containing 94% by weight of water for the 6% gel. The beads can beany convenient size and size range as are known and available in theart. The 4% and 6% gels available above typically comprise beads havinga diameter range between 60 and 120 microns.

These gel beads, in turn, can be utilized in several forms. For example,a "wet settled gel" is obtained when the gel beads in water are allowedto settle out under gravity, i.e., by draining off most of the water,leaving only the hydrated gel beads and interstitial water, i.e., waterbetween the beads. This typically results in a wet settled gelcomprising 70-80% hydrated bead volume and 20-30% interstitial watervolume, preferably about 75% by volume of hydrated beads about 25% byvolume of interstitial water. The wet settled gel form is convenient touse in processing because the material is mostly water providing adensity close to 1. This, in turn, provides flexibility in relativelyaccurate measuring either by weight or volume, especially when usinglarger quantities, i.e., 10 grams and above.

A "moist" or "sucked" gel comprises the hydrated gel beads with theinterstitial water removed and is a more accurate way to measure smalleramounts of gel.

In the processing discussed below and in the Examples which follow, theagarose gel, agarose gel beads, agarose or agarose beads refers to a wetsettled gel unless otherwise noted.

The aqueous composition of the present invention preferably comprisesavidin agarose gel beads (wet settled gel) in a slurry or suspensionwith a solution comprising:

1 to 50% by weight of the bulking agent;

0.01 to 50% by weight of the protectant; and

40 to 98.99% by weight of water.

More preferably, the aqueous composition according to the presentinvention comprises avidin agarose gel beads in a slurry or suspensionwith a solution comprising:

5 to 40% by weight and most preferably 10% by weight of a bulking agent,preferably a sugar, more preferably maltose;

0.1 to 10% by weight and most preferably 1% by weight of a protectant,preferably an antioxidant, more preferably ascorbic acid; and

50 to 94.9% by weight of water;

and optionally, in a preferred embodiment, further including:

an agent sufficient to adjust the pH to a desired level, preferably analkaline material, e.g., sodium hydroxide to adjust the pH to about 4;and

a buffer, which is preferably the ascorbic acid protectant.

Typically the slurry or suspension comprises about 10 to about 70% byvolume of wet settled gel beads in about 30 to 90% of one of the above"protectant" solutions, it being understood that compositions having 10%beads are in a suspension whereas those compositions having 70% beadsare in the form of a slurry or even a gel.

In order to immobilize the avidin to a support, e.g., agarose, thesupport must be pre-activated prior to avidin coupling. A preferredprocess involves the use of epichlorohydrin as the activating agent,however, activation can be carried out by any suitable technique capableof providing an activated support which can form covalent bonds withavidin. For example, various activation reagents available forderivatizing supports are: diazonium groups, isocyanate groups, acidchloride groups, acid anhydride groups, sulfonyl chloride groups,dinitro fluorophenyl groups, isothiocyanate groups, hydroxyl groups,amino groups, n-hydroxysuccinmide groups, triazine groups, hydrazidegroups, carbodiimide groups, silane groups, aldehydes, 1, 4-butanedioldiglycidyl ether, sodium metaperiodate, 1, 1-carbonyl diimidazole,divinylsulphone, 2-fluoro-1-methylpyridinium toluene-4-sulphonate andcyanogen bromide. See (a) Pentapharm Patent DT 2440 254 A1; (b) P. D. G.Dean, W. S. Johnson and F. A. Middle (Editors) (1991) IRL PressOxford--Affinity Chromatography--A practical approach--chapter2--Activation Procedures and (c) C. R. Lowe and P. D. G. Dean (1974)John Wiley and Sons Ltd., London, Affinity Chromatography, thedisclosures of which are incorporated herein by reference.

The preferred activation chemistry is by means of an epoxide groupfollowing activation with epichlorohydrin. The use of a supportactivated in this manner results in essentially no avidin leaching afteravidin bonding.

Generally, the support is activated by a highly reactive compound, whichsubsequently reacts with a functional group of the ligand, e.g., --OH,--NH₂, --SH, --CHO, to form a covalent linkage. Remaining active groups,which have no avidin attached, can be, but it is not essential, blockedwith compounds such as ethanolamine, acetic anhydride or glycine.

The preferred activation chemistries for use in the subject matterinvention are:

(a) Activation of the support by epichlorohydrin or a bifunctionalepoxide compound followed by coupling avidin via --NH₂, --SH or --OHgroups.

(b) Cyanogen bromide activation followed by direct coupling of avidinvia --NH₂ groups on the protein.

(c) Activation of the support with monochlorotriazine followed bycoupling of avidin via --NH₂, --OH or --SH groups.

(d) Activation of the support with dichlorotriazine followed by couplingof avidin via --NH₂, --OH or --SH groups.

(e) Tresyl chloride activation of the support followed by coupling ofavidin via --NH₂, --OH or --SH groups.

(f) Activation of the support with adipic acid hydrazide or hydrazidefollowed by coupling of oxidized avidin via --CHO groups.

(g) Activation of the support with an amino ligand followed by couplingof oxidized avidin via --CHO groups.

All the above preferred methodologies employ agarose as the support,however, it is possible to use other aforementioned supports as well.For example, when using silica, the preferred activation chemistriesare:

(a) Activation of the support by epichlorohydrin or a bifunctionalepoxide compound followed by coupling avidin via --NH₂, --SH or --OHgroups.

(b) Gamma--glycidoxypropyltrimethoxysilane activation with directcoupling of the avidin via --NH₂ groups on the protein.

(c) Cyanogen bromide activation followed by direct coupling of avidinvia --NH₂ groups on the protein.

(d) Gamma--glycidoxytrimethoxysilane activation followed by opening ofthe epoxide ring to form a diol group, which can be subsequentlyactivated with cyanogen bromide. Direct coupling of the avidin can beachieved via --NH₂ groups on the protein.

(e) Gamma--glycidoxypropyltrimethoxysilane activation followed bypreparation of amino-silica by treatment with ammonia solution.

The amino-silica can be subsequently activated with cyanuric chloride(triazine) and the avidin coupled via --NH₂, --OH or --SH groups.

Coupling of the avidin to the activated support must be buffered at acertain pH to obtain optimal avidin binding. Generally, with standardactivation techniques such as gamma--glycidoxypropyltrimethoxysilanecoupling of avidin to activated support and cyanogen bromide coupling ofany protein to active groups requires buffering at a pH 1-2 units higherthan the pKa of the primary and secondary amines of the avidin. However,the use of cyanuric chloride as the activator enables the use of muchlower pH buffers (optimal coupling pH is 4-6). Another method ofcoupling avidin to an inert support is via its carbohydrate moieties.This involves first the oxidation of the sugar group to --CHO groupsfollowed by direct coupling a acid pH to an amino group such ashydrazide. A wide range of coupling buffers can be used. See, forexample, Table 1.

                                      TABLE 1                                     __________________________________________________________________________    EXAMPLES OF COUPLING BUFFERS USED IN AVIDIN IMMOBILIZATION                    TO SILICA AND AGAROSE SUPPORTS                                                SUPPORT                                                                            ACTIVATION METHOD                                                                              COUPLING BUFFER                                         __________________________________________________________________________    Silica                                                                             gammaglycidoxypropyltrimethoxysilane                                                           0.1M Sodium bicarbonate pH 8-9 10mM                                           HEPES pH 7.0                                            Silica                                                                             Y-glycidoxypropyltrimethoxysaline +                                                            0.1M Sodium bicarbonate pH 8-9 10mM                          cyanogen bromide HEPES pH 7.0                                            Silica                                                                             Cyanogen Bromide Water pH 7.0 0.1M Sodium bi-                                                  carbonate pH 7-9 10mM HEPES pH 7.0                      Agarose                                                                            Monochlorotriazine                                                                             50mM Sodium Acetate/1MNaC1 pH 4.0                       Agarose                                                                            Dichlorotriazine 0.1M Potassium phosphate/1MNaCl pH                                            8.0-9.0                                                 Agarose                                                                            Tresyl chloride  50mM Potassium phosphate/0.5M NaCl                                            pH 7.7                                                  Agarose                                                                            Hydrazide        50mM Sodium Acetate pH 5.5 10mnM                                              NaBH.sub.4                                              Agarose                                                                            Amine            50mnM Sodium Acetate pH 5.5 10mM                                              NaBH.sub.4                                              Agarose                                                                            Epoxide          20mM Sodium Bicarbonate/0.5M NaCl                                             pH 10.0                                                 __________________________________________________________________________

As described previously, these "high activity" compositions of avidinimmobilized on an inert support, e.g., agarose, are useful in any andall chemical and biological applications where present avidin technologyis useful. In a preferred embodiment, the avidin immobilized ontoagarose is thereafter incorporated into composition of this inventionconveniently by mixing the various components in water or by mixing the"protectant" solution components in water and thereafter adding theavidin agarose gel. For situations requiring a sterile aqueouscomposition this can be carried out aseptically or preservatives can beadded to the composition. Preferably, the aqueous composition islyophilized into a powder form which can be terminally sterilized, e.g.,by gamma irradiation. Any convenient freeze-drying process can beemployed. A preferred process involves cooling the aqueous compositionin a lyophilization apparatus to about -33° C. and maintaining thiswhile a vacuum is initiated and the composition is dried under a reducedpressure of about 0.3 mbar. Thereafter, the composition is allowed towarm to room temperature.

The compositions of this invention involving the use of stable avidincompositions for the capture of a biotinylated form of thrombin or athrombin-like enzyme, e.g., Batroxobin, are useful in methods to convertfibrinogen, or a fibrinogen-containing composition, into fibrin monomer,or a fibrin monomer-containing composition. Accordingly, the presentinvention further includes a novel method, to prepare a fibrin monomeruseful, for example, in preparing a fibrin sealant. This novel methodinvolves

subjecting a source of fibrinogen to a biotinylated thrombin orthrombin-like enzyme composition to convert fibrinogen into fibrinmonomer,

"capturing" the biotinylated enzyme with an avidin composition of thisinvention to form a biotin/avidin complex, and

removing the enzyme which is a part of the so-formed biotin/avidincomplex.

The compositions of the present invention can further be incorporatedinto a processing unit, e.g., an automated centrifuge for preparingfibrin monomer as defined above. The avidin agarose composition can bepreloaded into the processing unit in powder form or can be lyophilizedin situ in the device or in a controlled release compartment of thedevice.

EXAMPLE 1 AQUEOUS COMPOSITION

Approximately 3.5 liters of beaded agarose gel (grade 4XL commerciallyavailable as Sepharose CL-4B™ from Pharmacia Co.) was gravity settled ina filter funnel. Approximately 2.8 liters of the so-settled agarose gelwas transferred to a reaction vessel. The agarose gel was washed 12times with 2.8 liter volumes of water. The so-washed gel was thereaftermixed with 2016 milliliters of 0-11 Molar sodium hydroxide and thenreacted with 202 milliliters of epichlorohydrin for about 3 hours whilemaintaining 40° C. This activated gel was then washed with water andthereafter coupled to 19.6 grams of avidin in the presence of a sodiumchloride/sodium bicarbonate pH 10 buffer at about 40° C. for 48 hours.The avidin-agarose gel was thereafter washed several times with sodiumchloride solution and any unreacted epoxide groups were blocked bytreatment with 1M ethanolamine (pH 9.5) for 16 hours at 20° C. Theavidin-agarose gel was next washed with water and thereafter mixed withan equal volume (28L) of a solution containing maltose (20% w/v) andascorbic acid (2% w/v) at pH 4.0 and allowed to drain under gravity.

EXAMPLE 2 LYOPHILIZED COMPOSITION

The end-product provided by the method of Example 1, above, was placedon trays and loaded into a EF6(S) lyophilization apparatus (availablefrom Edwards High Vacuum Co.) in which the shelves had been pre-cooledto -37° C. The avidin agarose slurry was cooled to -33° C. and thepressure was reduced (by vacuum) to 0.3 millibars. The product wasmaintained at this pressure and temperature until all of the ice hadsublimed (about 70 hours). The pressure was then adjusted to 0.08millibars and the temperature was raised stepwise 5° C. per hour to 30°C. to provide the lyophilized product.

We claim:
 1. A solid or powder composition comprising:an inert supportmaterial; and, avidin immobilized on said inert support material whereinsaid composition has at least 1000 biotin binding units of activity pergram of the composition.
 2. The composition of claim 1 having between1500 and 3000 units of biotin binding activity per gram of thecomposition.
 3. The composition of claim 1 having between 1800 and 2400units of biotin binding activity per gram of the composition.